Project Summary Macrophages as a key cell type in the innate immune system are involved in a wide spectrum of allograft responses, but exactly what they do and by what mechanisms they affect diverse allograft outcomes remains poorly understood. We recently reported the identification of Paired Ig-like Receptors (PIR) as an innate allorecognition system that allows myeloid cells to acquire features of allospecificity and innate memory in transplant models (Science 2020;368:1122), thus uncovering new opportunities in better understanding and potentially targeting those cells in favor of transplant survival. However, outstanding questions remain regarding the longevity of innate memory macrophages in vivo, how PIR-A/B receptors are regulated, and the exact identity of macrophages in tolerized grafts. Now we have generated new preliminary data showing that the chromatin structures at Pira and Pirb loci are strikingly different and that they are controlled by distinct epigenetic mechanisms downstream of CD40 and PIR-A3 signaling pathways. Furthermore, the anti-donor innate memory macrophages in vivo are incredibly stable as a result of the concerted actions of CD40 and PIR-A3. The goal of this project is to dissect mechanistically how the anti-donor innate memory macrophages are induced and maintained long-term, testing the hypothesis that the “open” chromatin modifications at the Pira3 locus downstream of CD40 signaling pathways mediate PIR-A3 expression via allelic exclusion and that the subsequent PIR-A3 signaling then programs stable memory features of innate macrophages upon binding to MHC-I. We put together 3 Aims to test this hypothesis. Aim 1 is to determine whether induction of anti-donor innate macrophage memory depends on “open” chromatin remodeling at Pira3 locus downstream of CD40 signaling as well as the NF-kB pathways involved (canonical vs. non-canonical). Aim 2 is to examine whether the long-term maintenance of anti- donor innate memory macrophages requires PIR-A3 signaling, especially in driving epigenetic and metabolic rewiring and cell survival. Aim 3 is to use a mouse heart transplant model to test whether decommissioning the anti-donor innate memory macrophages is a fundamental mechanism of tolerance induction upon targeting the CD40/PIR-A3 axis. We believe that studies in this application are addressing a significant knowledge gap in transplantation, and the animal models, tools, and multi-omics approaches we have developed in the lab put us in a unique position to carry out the proposed studies and move the field forward.